INSECTICIDE DETOXIFICATION IN THE NAVEL ORANGEWORM

INSECTICIDE DETOXIFICATION IN THE NAVEL ORANGEWORM AMYELOIS

TRANSITELLA (LEPIDOPTERA: PYRALIDAE)

BY

MARK R. DEMKOVICH

THESIS

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Entomology

in the Graduate College of the

University of Illinois at Urbana-Champaign, 2014

Urbana, Illinois

Master’s Committee:

Professor May R. Berenbaum, Chair

Professor Mary A. Schuler

Professor Hugh M. Robertson

ii

ABSTRACT

The polyphagous navel orangeworm (Amyelois transitella) is considered the most

destructive pest of introduced nut crops, including almonds and pistachios, in California

orchards. Management of this pest has typically been a combination of cultural controls

(including removal of unharvested fruits) and the use of insecticides; insecticide use has

increased substantially along with the value of these commodities. In a series of dietary studies

the effects of the cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO)

and the glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) were assessed on the

toxicity of the insecticides azinphos-methyl, chlorpyrifos, chlorantraniliprole, β-cyfluthrin, and

bifenthrin to first instar A. transitella larvae from a laboratory strain. Piperonyl butoxide

interacted antagonistically with the organophosphate insecticides azinphos-methyl and

chlorpyrifos, indicating that they likely are bioactivated by P450s. Piperonyl butoxide synergized

the toxicity of the pyrethroids β-cyfluthrin and bifenthrin, which suggests that P450s are

involved in detoxification of pyrethroid insecticides. Only azinphos-methyl was a substrate for

GSTs in A. transitella, as evidenced by diethyl maleate synergism assays. Neither PBO nor DEM

influenced the toxicity of the anthranilic diamide chlorantraniliprole. Results suggest that if A.

transitella detoxify other classes of insecticides used in management through enhanced P450

activity, and resistance begins to develop by this route, then incorporating organophosphate

insecticides into rotations may provide an effective means of prolonging efficacy of chemical

control because their speed of activation will increase.

In a related series of studies to characterize a putatively pyrethroid-resistant strain of

navel orangeworm, eggs from adults originating from almond orchards in which pesticide

failures were reported in Kern County, California were shipped to the University of Illinois at

iii

Urbana-Champaign. Their susceptibility to bifenthrin and β-cyfluthrin was compared to that of

an established colony of navel orangeworms. Administration of piperonyl butoxide and S,S,S-

tributyl phosphorotrithioate (DEF) in bioassays with the pyrethroids bifenthrin and β-cyfluthrin

produced synergistic effects and demonstrated that P450s and carboxylesterases (COEs)

contribute to resistance in this navel orangeworm population. Resistance is therefore primarily

metabolic and likely the result of overexpression of specific P450 and COE genes. Bioassays

involving pesticides routinely used in tank mixtures indicate that chlorantraniliprole, which is not

detoxified by P450s, may be more effective than methoxyfenozide in overcoming existing

pyrethroid resistance because chlorantraniliprole enhanced pyrethroid toxicity and

methoxyfenozide had no effect.

Results from median-lethal concentration (LC50) assays revealed that resistance was

maintained across eight generations in the laboratory. Life history trait comparisons between the

resistant strain and susceptible strain revealed significantly lower pupal weights in resistant

males and females reared on the same wheat bran-based artificial diet across three generations.

The number of days until the first molt was significantly greater in the resistant strain than the

susceptible strain, although overall development time was not significantly different between

strains. These experiments indicate that resistance is heritable and may have an associated fitness

cost, which could influence the dispersal and expansion of resistant populations.

iv

Acknowledgments

I would like to express my gratitude to my advisor Dr. May Berenbaum for her

encouragement and support throughout this project and to Dr. Mary Schuler for her continuous

support and guidance throughout my undergraduate and graduate career. I also thank Dr. Hugh

Robertson for offering his time and expertise toward the navel orangeworm. I would like to

thank all current members of the Berenbaum laboratory who assisted me in any aspect of this

project. I thank Katherine Noble for introducing me to the world of navel orangeworms and for

the guidance I received as I began working with this wonderful insect pest. I thank Dr. Joel

Siegel for providing expertise and encouragement as each stage of this project advanced. I owe

one final thank you to my roommate and good friend Sam Oehlert for his amazing technical

support during the construction of this thesis. Funding was provided by the Almond Board of

California and the California Pistachio Research Board.

v

TABLE OF CONTENTS CHAPTER I: NAVEL ORANGEWORM DETOXIFICATION MECHANISMS FOR

DIFFERENT CLASSES OF INSECTICIDES CURRENTLY USED IN MANAGEMENT ........1

CHAPTER II: MECHANISM OF RESISTANCE ACQUISITION IN NAVEL

ORANGEWORMS EXPOSED TO PYRETHROID INSECTICIDES ........................................27

CHAPTER III: LIFE HISTORY DIFFERENCES BETWEEN A RESISTANT AND

SUSCEPTIBLE STRAIN OF NAVEL ORANGEWORMS .........................................................51

1

CHAPTER I: NAVEL ORANGEWORM DETOXIFICATION MECHANISMS FOR

DIFFERENT CLASSES OF INSECTICIDES CURRENTLY USED IN MANAGEMENT

Introduction

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is

considered the most destructive pest of introduced nut crops in California orchards (Connell

2001; Bentley et al. 2008; Zalom et al. 2012). The geographic range of the navel orangeworm

extends from the southern tier of the United States, through Mexico and Central America, and

into South America (Heinrich 1956). Larval hosts within its native range belong to diverse plant

families, including Asparagaceae, Fabaceae, Rubiaceae and Sapindaceae (Heinrich, 1956).

Although this insect pest was initially described feeding on fallen fruits of Citrus sinensis

(Rutaceae), the introduction of additional nonindigenous fruit crops allowed the navel

orangeworm to expand its range and establish itself as a pest in California orchards by the 1940s

(Wade 1961). The navel orangeworm causes economic damage to almonds (Rosaeae), pistachios

(Anacardiaceae), figs (Moraceae), pomegranates (Lythraceae), and walnuts (Juglandaceae) in the

Central Valley of California, demonstrating its capacity to consume host plants with distinct

chemistries (Bentley et al. 2008).

Navel orangeworm is an internal feeder with a preference for fallen and mummy

(unharvested) fruit or injured and diseased fruits (Heinrich 1956) Neonates tunnel into nuts,

resulting in consumption of the nutmeat and the production of large quantities of frass and

webbing. Moreover, infestation by navel orangeworm results in an increased susceptibility to

infection by Aspergillus species, many of which produce aflatoxins, a crop contaminant that

causes millions of dollars in losses each year (Campbell et al. 2003; Molyneux et al. 2007; van

2

Egmond et al. 2007). Management of this insect pest has typically been a combination of cultural

control (removal of unharvested fruits) combined with insecticides, but the use of insecticides

has increased substantially along with the value of these commodities.

The ability to feed on chemically distinct host plants may have preadapted the navel

orangeworm for the different types of xenobiotics it encounters in the Central Valley of

California, including synthetic and organic insecticides, mycotoxins, and phytochemicals

produced by host plants (Niu et al. 2012). Compared to other insects, navel orangeworm can

tolerate unusually high concentrations of aflatoxin (>100 g/g) administered in artificial diet

(Niu et al. 2009) as a result of an active cytochrome P450 monooxygenase (P450) system that

can convert aflatoxin B1 into less toxic metabolites (Lee and Campbell 2000). CYP6AB11 has

been characterized in the navel orangeworm as a P450 enzyme that can metabolize imperatorin,

a furanocoumarin with similar chemistry to other phytochemicals present in several navel

orangeworm host plants such as Citrus spp. and fig (Ficus carica) (Niu et al. 2011). The

existence of specialized P450s in the navel orangeworm may have facilitated the transition to

non-native crop plants because larvae could detoxify compounds produced by host species with

structurally similar phytochemicals.

P450s, GSTs, and carboxylesterases (COE) are three major detoxification enzyme

systems utilized in metabolism of pesticides by insects (Feyereisen 2005; Oakeshott et al. 2005;

Ranson and Hemingway 2005). Insecticides currently registered for use in California orchards

include members of the pyrethroid, organophosphate, anthranilic diamide, diacyl hydrazine,

neonicotinoid, and spinosyn classes of insecticides. Insecticides sprayed to control navel

orangeworms in heavily infested orchards are usually applied in rotation when the hulls split and

the kernel is exposed to larval feeding and infection by Aspergillus (Niu et al. 2012). Although

3

applicators may assume that the rotation of different insecticides with different modes of action,

applied either individually or in combination, will delay resistance acquisition, rotational patterns

may also raise the risk of acquisition of multiple resistance if rotated insecticides share a

common route of detoxification.

In this study, I examined the effects of two synergists, piperonyl butoxide (PBO) and

diethyl maleate (DEM), which inhibit detoxification of xenobiotics by P450s and GSTs,

respectively, on the toxicity of the insecticides azinphos-methyl, chlorpyrifos,

chlorantraniliprole, β-cyfluthrin, and bifenthrin. These insecticides represent member(s) of the

organophosphate, anthranilic diamide, and pyrethroid insecticide classes used to control the

navel orangeworm (Table 1.1). Synergism (enhancement of mortality beyond additive effects) in

the presence of one of these specific inhibitors implicates the contribution of the enzyme system

to metabolism of the insecticides assayed.

Materials and Methods

Chemicals:

Piperonyl butoxide was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Azinphos-

methyl, bifenthrin, β-cyfluthrin, chlorantraniliprole, chlorpyrifos, and diethyl maleate were

purchased from Sigma (St. Louis, MO). Bifenthrin was purchased from Chem Service, Inc.

(West Chester, PA). Bifenthrin and chlorpyrifos were dissolved in methanol. Azinphos-methyl,

β-cyfluthrin, and chlorantraniliprole were dissolved in acetone. All stock solutions were stored at

-20ºC.

Navel orangeworm colony:

A laboratory colony of A. transitella designated as SPIRL-1966 (Siegel et al. 2010) was

kept in the insectary at University of Illinois at Urbana-Champaign and maintained at conditions

4

of 27 ± 4ºC with a photoperiod of 16:8 (L:D) hours. Larvae were mass-reared until pupation in

500-ml glass jars containing 300 g of a wheat bran diet (Finney and Brinkmann 1967). Adults

were transferred to additional 500-ml glass jars with tissue paper on the inside and covering the

top to serve as oviposition substrate. Eggs were collected every 48 hours from these jars. First

instar larvae were selected within 24 hours of hatching. Four larvae were gently transferred into a

28-ml (1-oz) plastic cup containing 5 g of unaugmented (control) diet or diet mixed with

different concentrations of insecticide in the bioassays. A total of 20 larvae (5 cups) were

prepared for each treatment with or without insecticide application.

Insecticide Preparation:

Insecticides were incorporated into standard diet at specific concentrations and fed to

neonate larvae. Insecticides were mixed into the standard diet in its liquid phase and then poured

into separate 1-oz cups to harden. Solutions of each insecticide in methanol or acetone were

prepared at a range of concentrations (azinphos-methyl: 0.2, 0.5, 1, 2, 3, and 5 µg/g; β-cyfluthrin:

50 ng/g, 100 ng/g, 150 ng/g, 500 ng/g, and 1µ/g; bifenthrin: 50 ng/g, 200 ng/g, 300 ng/g, and 500

ng/g; chlorantraniliprole 0.4, 4, 6, 10, and 16 µg/g; chlorpyrifos: 0.05, 0.2, 0.3, 0.5, 1, and 2

µg/g). In these bioassays, 20 neonates were exposed to each concentration of insecticide.

Mortality levels were assessed and recorded after 48 hours with at least three replicates at each

concentration. Larvae that did not move when touched with a soft brush were scored as dead.

Synergist Preparation:

A concentration of 200 µg/g for piperonyl butoxide was previously established by Niu et

al. (2012) as the maximum concentration determined to be nonlethal for first instar larvae. For

diethyl maleate, a concentration of 200 µg/g produced <15% morality after 48 hours, and this

concentration was used in all assays.

5

Bioassays:

Insecticide assays were conducted using median-lethal concentrations for each

insecticide (1.6 µg/g azinphos-methyl; 40 ng/g β-cyfluthrin; 200 ng/g bifenthrin; 4 µg/g

chlorantraniliprole; 400 ng/g chlorpyrifos) mixed with standard insect diet in the presence or

absence of piperonyl butoxide (200 µg/g) or diethyl maleate (200 µg/g). Controls present in each

bioassay consisted of 200 µl insecticide solvent mixed into diet in the absence of insecticide and

200 µg/g of either PBO or DEM. Each bioassay exposed 20 first instar larvae to a single

concentration of insecticide. All bioassays were repeated four times. Mortality was recorded

daily until at least 80% of the treated insects were dead. On the rare occasions that cannibalism

or larval scavenging of already-dead individuals occurred, larvae were removed from the sample

size.

Statistical Analyses:

SPSS version 22 software (SPSS Inc., Chicago IL) was used to run the Probit analysis

and generate an estimated calculation of the median lethal concentration that would kill 50% of

the sample population at 48 hours (LC50) for the five insecticides. Separate analyses using JMP

Pro version 9 (SAS Institute, Cary NC) were calculated for each insecticide and insecticide with

synergist using multiple regression with orthogonal polynomial contrasts in order to differentiate

among treatments.

Results

Synergistic or antagonistic interactions were observed when the mortality of the

insecticide with synergist was significantly higher (P<0.05) than the combined mortality of the

insecticide and synergist. Antagonistic interactions were observed when the mortality of the

6

insecticide with synergist was significantly lower (P<0.05) than the combined mortality of the

insecticide and synergist.

The goodness-of-fit test indicated that insecticide concentration mortality data fit the

Probit model (P>0.05) for azinphos-methyl, chlorpyrifos, chlorantraniliprole, β-cyfluthrin and

bifenthrin (Table 1.2). Each class of insecticide displayed differential toxicity toward the navel

orangeworm after 48 hours. The pyrethroids β-cyfluthrin and bifenthrin, with LC50s at 40 ng/g

and 200 ng/g, respectively, were toxic to neonates at the lowest concentration of all insecticides

tested. The organophosphates azinphos-methyl and chlorpyrifos were lethal to neonates at higher

concentrations, with LC50s at 1.6 µg/g and 410 ng/g, respectively, and the anthranilic diamide

chlorantraniliprole was least toxic, with an LC50 of 4 g/g.

Mortality levels were significantly different (P<0.001) between insecticide treatments

and the controls across all time points examined for the organophosphates azinphos-methyl and

chlorpyrifos. The synergist and insecticide solvent controls were not significantly different from

each other (P>0.05) across each time point examined in these insecticides. Piperonyl butoxide

exhibited antagonistic interactions with both insecticides and significantly reduced the toxicity at

36 hours (P=0.014) through 144 hours (P<0.001) for azinphos-methyl (Fig. 1.1) and at 36 hours

(P=0.016) through 96 hours (P<0.001) for chlorpyrifos (Fig. 1.3) Diethyl maleate synergized the

toxicity of azinphos-methyl at 24 hours (P=0.043) through 96 hours (P=0.027) (Fig. 1.2) but had

no significant effect on chlorpyrifos toxicity from 24 hours (P=0.410) to 96 hours (P=0.057)

(Fig. 1.4).

Mortality levels were significantly different (P<0.001) between insecticide treatments

and the controls from 24 hours to 120 hours in chlorantraniliprole assays. The synergist and

insecticide solvent controls were not significantly different from each other (P>0.05) from 24

7

hours to 120 hours in assays with PBO and DEM. Piperonyl butoxide had no significant effect

on the toxicity of chlorantraniliprole from 24 hours (P=0.816) to 120 hours (P=0.660) (Fig. 1.5).

Diethyl maleate had no significant effect on the toxicity of chlorantraniliprole from 24 hours

(P=0.629) to 120 hours (P=0.659) (Fig. 1.6).

A significant difference in mortality levels (P<0.001) was observed between insecticide

treatments and the controls across each time point examined in the pyrethroids β-cyfluthrin and

bifenthrin. The synergist and insecticide solvent controls were not significantly different from

each other (P>0.05) across each time point examined in these pyrethroids. Piperonyl butoxide

synergized the toxicity of β-cyfluthrin at 24 hours (P<0.001) through 144 hours (P<0.001)

(Fig.1.7) and the toxicity of bifenthrin at 24 hours (P<0.001) through 144 hours (P<0.001) (Fig.

1.9). Diethyl maleate had no significant effect on the toxicity of β-cyfluthrin from 24 hours

(P=0.396) to 144 hours (P=0.060) (Fig. 1.8) or the toxicity of bifenthrin from 24 hours

(P=0.320) to 144 hours (P=0.356) (Fig. 1.10).

Discussion

There are currently numerous insecticides registered for use to control lepidopteran and

hemipteran pests in California orchards (Niu et al. 2012). In pistachio orchards, buprofenzin

(Applaud) and neonicotinoid insecticides are used to control the mealybug (Ferrisia gilli)

(Hemiptera: Pseudococcidae) and are applied when navel orangeworms are present (Haviland et

al. 2012). Peach twig borers (Anarsia lineatella) (Lepidoptera: Gelechiidae) feed on new crop

nuts following hull split, and infestations in almond orchards can result in navel orangeworm

exposure to additional insecticides because navel orangeworms prefer damaged nuts and are

likely to invade any that were previously damaged by peach twig borers (Zalom et al. 2002;

Hamby and Zalom 2013). This variation in chemical control strategies for other insect pests

8

means that navel orangeworms may be exposed to as many as four classes of insecticide in a

single season (Niu et al. 2012). The rapid expansion of almond and pistachio orchards over the

past seven years into orchards adjacent to cotton, grapes, and stone fruits has increased the

probability of nontarget exposure of navel orangeworms to insecticides as a result of drift, which

may increase selection pressure on the navel orangeworm for resistance to multiple classes of

insecticide (Higbee and Siegel 2009).

Although P450s generally metabolize xenobiotics into less toxic metabolites, they may

bioactivate certain compounds instead, resulting in a metabolite more toxic than the original

compound (Scott 1999; Wegorek et al. 2011). The results from assays involving chlorpyrifos and

azinphos-methyl indicate that both insecticides are bioactivated by PBO in the navel

orangeworm because mortality decreased in the presence of the P450 inhibitor. Ahmad and

Hollingsworth (2004) observed an increased tolerance to azinphos-methyl and chlorpyrifos when

PBO was applied in bioassays involving second instar obliquebanded leafrollers (Choristoneura

rosaceana) (Lepidoptera: Tortricidae), indicating that P450-mediated bioactivation of these

organophosphates occurs in this lepidopteran pest. An increase in survivorship was observed in

navel orangeworm bioassays because inhibition of P450s likely prevented the conversion of the

organophosphate to the toxic oxon form and subsequent binding to acetylcholinesterase.

Bioassays involving the organophosphates and diethyl maleate indicated that azinphos-

methyl was metabolized by GSTs and chlorpyrifos was not. Enhanced GST activity has been

established as a mechanism of resistance to organophosphates in populations of lepidopteran

pests such as the codling moth (Cydia pomonella) (Lepidoptera: Tortricidae) and the

obliquebanded leaf roller (Smirle et al. 1998; Ahmad and Hollingsworth 2004; Fuentes-

Contreras et al. 2007). The primary mode of detoxification of chlorpyrifos in the navel

9

orangeworm remains to be investigated. Further work is clearly warranted to delineate the effects

of the esterases in detoxification of these organophosphate insecticides.

Pyrethroids are the most widely used insecticide in navel orangeworm management

during the growing season (Leal et al. 2009). The primary modes of detoxification for pyrethroid

insecticides include P450s and esterases (Ishaaya 1993). The toxicity of both β-cyfluthrin and

bifenthrin was enhanced in the presence of PBO, indicating that P450s are involved in the

metabolism of pyrethroids in this species. Niu et al. (2012) examined neonate mortality in navel

orangeworms when piperonyl butoxide was applied to median-lethal concentrations of the

pyrethroids α-cypermethrin and τ-fluvalinate and found that both pyrethroids exhibited

synergistic effects with PBO. The findings from these experiments emphasize the participation

of P450s in the metabolism of pyrethroids and suggest that disruption of P450 enzyme systems

could increase the efficacy of this insecticide class; however, pyrethroids used to manage other

insect pests may increase selection pressure on navel orangeworms and lead to the development

of resistance if applied in close proximity to almond and pistachio orchards (Niu et al. 2012).

Diethyl maleate did not have any significant impact on the metabolism of β-cyfluthrin or

bifenthrin in navel orangeworms. This is consistent with findings of previous researchers that

GSTs were not linked to the direct metabolism of pyrethroids (Enayati et al. 2005; Khambay and

Jewess 2005; Ranson and Hemingway 2005). Although results from this experiment support

P450-mediated metabolism as major detoxification pathways in the navel orangeworm, the full

extent of GST involvement in metabolism of pyrethroids remains unclear. GST-dependent

sequestration of pyrethroids and detoxification of pyrethroid-induced lipid peroxidation products

have been documented as potential resistance mechanisms in other insects such as the mealworm

10

(Tenebrio moliter) (Coleoptera: Tenebrionidae) and the brown planthopper (Nilaparvata lugens)

(Hemiptera: Delphacidae) (Kostaropoulos et al. 2001; Vontas et al. 2001).

A recent addition to the arsenal of chemicals used in navel orangeworm management is

chlorantraniliprole, an anthranilic diamide insecticide with broad-spectrum activity against

lepidopteran pests because of its activity on the calcium channels of muscle (Temple et al. 2009).

There was no evidence of synergism when PBO and DEM were applied to chlorantraniliprole in

bioassays with the navel orangeworm. Wang et al. (2010) reported that PBO and DEF displayed

minor synergism with a susceptible strain in the diamondback moth (Plutella xylostella)

(Lepidoptera: Plutellidae), but PBO, DEM, and DEF had no activity in field populations of this

insect. It is possible that P450s and GSTs may not be the main detoxification systems for

chlorantraniliprole in the navel orangeworm. Tolerance may be attributable to esterase activity

or, alternatively, to varying degrees of target-site insensitivity. The contribution of esterases to

chlorantraniliprole will be evaluated in future assays.

Disruption of P450 enzymes responsible for the metabolism of many insecticides (Lee

and Campbell 2000; Niu et al. 2012) may be a valid strategy for navel orangeworm control. If

the navel orangeworm detoxifies other classes of insecticides through enhanced cytochrome

P450 enzyme activity and resistance begins to develop by this route, then incorporating

organophosphates into insecticide rotation may provide an effective system for delaying

resistance to chemical control. This study demonstrated that chlorantraniliprole is not detoxified

by P450s or GSTs, which may allow it to be combined with other insecticide classes that are

detoxified by these systems, such as pyrethroids, in order to delay resistance acquisition. It is

essential to investigate the role of esterase genes in the detoxification of the different insecticide

classes that are used in management of navel orangeworms in order to avoid cross-resistance to

11

insecticides that share the same modes of detoxification. Understanding how detoxification

enzymes respond to selective pressures exerted by different insecticides is critical in order to

design sustainable control strategies and minimize the evolution of resistance.

12

Figures and Tables

Figure 1.1. First instar navel orangeworm mortality across multiple time points following dietary

exposure to 1.6 µg/g azinphos-methyl, 1.6 µg/g azinphos-methyl + 200 µg/g piperonyl butoxide

(PBO), 200 µg/g PBO, and 200 µl acetone. Error bars represent the standard error. Letters A, B,

and C represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96 120 144

Mo

rta

lity

Hours After Exposure

Acetone

PBO

Azinphos-methyl

Azinphos-methyl

+PBO

A

B

C

C

13


exposure to 1.6 µg/g azinphos-methyl, 1.6 µg/g azinphos-methyl + 200 µg/g diethyl maleate

(DEM), 200 µg/g DEM, and 200 µl acetone. Error bars represent the standard error. Letters A, B,

and C represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96

Mo

rta

lity


Acetone

DEM

Azinphos-methyl

Azinphos-methyl

+DEM

A

B

C C

14


exposure to 400 ng/g chlorpyrifos, 400 ng/g chlorpyrifos + 200 µg/g piperonyl butoxide (PBO),

200 µg/g PBO, and 200 µl methanol. Error bars represent the standard error. Letters A, B, and C

represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96

Mo

rta

lity


Methanol

PBO

Chlorpyrifos

Chlorpyrifos + PBO

A

B

C

C

15


exposure to 400 ng/g chlorpyrifos, 400 ng/g chlorpyrifos + 200 µg/g diethyl maleate (DEM), 200

µg/g DEM, and 200 µl methanol. Error bars represent the standard error. Letters A and B


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96

Mo

rta

lity


Methanol

DEM

Chlorpyrifos

Chlorpyrifos + DEM

A

A

B

B

16


exposure to 4 µg/g chlorantraniliprole, 4 µg/g chlorantraniliprole + 200 µg/g piperonyl butoxide

(PBO), 200 µg/g PBO, and 200 µl acetone. Error bars represent the standard error. Letters A and

B represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96 120

Mo

rta

lity


Acetone

PBO

Chlorantraniliprole

Chlorantraniliprole +

PBO

A

A

B

B

17


exposure to 4 µg/g chlorantraniliprole, 4 µg/g chlorantraniliprole + 200 µg/g diethyl maleate

(DEM), 200 µg/g DEM, and 200 µl acetone. Error bars represent the standard error. Letters A

and B represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 12 24 36 48 72 96 120

Mo

rta

lity


Acetone

DEM

Chlorantraniliprole

Chlorantraniliprole +

DEM

A A

B B

18


exposure to 40 ng/g β-cyfluthrin, 40 ng/g β-cyfluthrin + 200 µg/g piperonyl butoxide (PBO), 200

µg/g PBO, and 200 µl acetone. Error bars represent the standard error. Letters A, B, and C


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Acetone

PBO

β-cyfluthrin

β-cyfluthrin + PBO

A

B

C C

19


exposure to 40 ng/g β-cyfluthrin, 40 ng/g β-cyfluthrin + 200 µg/g diethyl maleate (DEM), 200

µg/g DEM, and 200 µl acetone. Error bars represent the standard error. Letters A and B represent

significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Acetone

DEM

β-cyfluthrin

β-cyfluthrin + DEM

A

A

B B

20


exposure to 200 ng/g bifenthrin, 200 ng/g bifenthrin + 200 µg/g piperonyl butoxide (PBO), 200

µg/g PBO, and 200 µl methanol. Error bars represent the standard error. Letters A, B, and C


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Methanol

PBO

Bifenthrin

Bifenthrin + PBO

A

B

C

C

21

Figure 1.10. First instar navel orangeworm mortality across multiple time points following

dietary exposure to 200 ng/g bifenthrin, 200 ng/g bifenthrin + 200 µg/g diethyl maleate (DEM),

200 µg/g DEM, and 200 µl methanol. Error bars represent the standard error. Letters A and B


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Methanol

DEM

Bifenthrin

Bifenthrin + DEM

A

A

B B

22

Table 1.1. Insecticides and synergists tested against first instar larvae from a laboratory strain

(SPIRL-1966) of Amyelois transitella.

Table 1.2. Probit analysis data for azinphos-methyl, chlorpyrifos, chlorantraniliprole, β-

cyfluthrin, and bifenthrin in neonate A. transitella from a laboratory strain (SPIRL-1966).

Insecticide n Slope (SE) LC50 ± 95% CL (µg/g) x2 P

Azinphos-methyl 636 2.73 (0.19) 1.50 (1.35-1.65) 6.22 0.40

Chlorpyrifos 555 4.20 (0.33) 0.41 (0.38-0.45) 7.49 0.19

Chlorantraniliprole 320 1.10 (0.33) 3.20 (0.92-4.83) 0.76 0.68

β-cyfluthrin 355 1.33 (0.22) 0.03 (0.01-0.05) 5.65 0.23

Bifenthrin 240 2.82 (0.35) 0.21 (0.18-0.25) 0.31 0.86

Active Ingredient Commercial

Name(s) Chemical Family Mode of Action

Dosage

used (µg/g)

Azinphos-methyl Guthion Organophosphate Acetylcholinesterase inhibitor 1.60

β-cyfluthrin Baythroid Pyrethroid Sodium channel modulators 0.04

Bifenthrin Brigade

Pyrethroid Sodium channel modulators 0.20

Bifenture

Chlorantraniliprole Altacor

Coragen Anthranilic diamide

Ryanodine receptor

modulators 4.00

Chlorpyrifos Lorsban Organophosphate Acetylcholinesterase inhibitor 0.40

Diethyl Maleate

Synergist Glutathione-S-transferase

inhibitor 200

Piperonyl Butoxide Butacide Synergist Cytochrome P450

monooxygenase inhibitor 200

23

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mycotoxins to navel orangeworm (Amyelois transitella) and corn earworm (Helicoverpa

zea). J. Chem. Ecol. 35: 951–957.

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selection and relationship to detoxication enzyme activity. Pest. Biochem. Physiol. 61:

183–189.

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defence agents confer pyrethroid resistance in Nilaparvata lugens. Biochem. J. 357: 65–

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Wade, W.H. (1961) Biology of the navel orangeworm, Paramyelois transitella (Walker), on

almonds and walnuts in northern California. Hilgardia 31, 129–171.

Wang, X., Li, X., Shen, A., and Wu, Y. (2010) Baseline susceptibility of diamondback moth

(Lepidoptera: Plutellidae) to chlorantraniliprole in China. J. Econ. Entomol. 103: 843–

848.

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Anarsia lineatella (Zeller), to organophosphate and pyrethroid insecticides. – Acta

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Guidelines: Almond. University of California ANR Publication 3431.

27

CHAPTER II: MECHANISM OF RESISTANCE ACQUISITION IN NAVEL

ORANGEWORMS EXPOSED TO PYRETHROID INSECTICIDES

Introduction

The ability of insects to develop resistance to synthetic insecticides presents a challenge

for sustainable pest management programs. Selection pressure from repeated exposure to

insecticides favors individuals with biochemical mechanisms that confer resistance by increasing

the rate at which detoxification occurs or that decrease overall sensitivity toward insecticides as a

result of changes in target site structure (Li et al. 2007). If resistance is heritable, then genes

encoding these mechanisms will be passed on to successive generations, resulting in populations

that cannot be controlled effectively through insecticide use (Khambay and Jewess 2005).

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is a

polyphagous insect pest that is able to feed on a diversity of chemically distinct and

economically important host plants, including almonds, pistachios, figs, walnuts, pomegranates,

and citrus (Connell 2001; Bentley et al. 2008; Zalom et al. 2012). The ability of the navel

orangeworm to cope with toxins produced from such divergent plant families suggests a versatile

detoxification system that may be preadapted for the acquisition of insecticide resistance (Li et

al. 2007).

Navel orangeworm resistance to standard applications of the pyrethroid insecticide

bifenthrin has recently been reported in almond orchards in Kern Country, California (B. Higbee,

Paramount Farming Company, personal communication). Many factors, such as the type of crop

and history of insecticide use, can affect selection pressure and lead to resistance within an insect

species (Gunning et al. 1991). Kern County had the largest acreage in production of almond and

pistachio crops from 2011-2012, with 20.1% of almond acres (148,913) in 2012 and 41.8% of

28

pistachio acres (62,831) in 2011 (ACP 2011; NASS 2012). Row crops grown adjacent to almond

and pistachio orchards, such as pomegranates and figs, may provide sources of immigrants when

infested by navel orangeworm (Burks et al. 2008). Chemical control practices that target navel

orangeworms and other insect pests within almond and pistachio orchards as well as neighboring

crops may increase selection pressure and promote resistance as a result of both movement

between hosts and drift exposure. Consequently, the navel orangeworm may be exposed to as

many as four insecticide classes within a single growing season and multiple insecticides within

each class (Niu et al. 2012).

The primary mechanisms involved in the generation of insecticide resistance to

pyrethroids involve target-site insensitivity, increased metabolism, or a combination of both

(Khambay and Jewess 2005; Li et al. 2007; Feyereisen 2011). Target-site resistance to

pyrethroids occurs through the inheritance of point mutations in the para-type sodium channels,

which are the binding sites for this insecticide class (Casida et al. 1983; Davies et al. 2007).

Metabolic resistance often occurs through contributions of cytochrome P450 monooxygenases

(P450s), glutathione-S-transferase (GSTs), and carboxylesterase (COEs) enzymes (Feyereisen

2005; Oakeshott et al. 2005; Ranson and Hemingway 2005; Liu 2012) and results in a decrease

in the effective dose of insecticide available at the target site.

Pyrethroids registered for use in almond and pistachio orchards include β cyfluthrin,

bifenthrin, esfenvalerate, fenpropathrin, lambda-cyhalothrin, and permethrin (CPRB 2012).

Pyrethroids mixed with diamides (e.g., chlorantraniliprole-Altacor; flubendiamide-Belt) or diacyl

hydrazines (e.g., methoxyfenozide-Intrepid) are also applied as tank mixes or as a premix

(Volium Xpress) in almonds and pistachios. Applicators assume that the rotation of different

insecticides with different modes of action, applied either individually or in combination, will

29

delay resistance acquisition but applying mixtures may also raise the risk of acquisition of cross-

resistance if the insecticides applied in mixtures share a common route of detoxification.

A compelling form of evidence for the contribution of an enzyme system in resistance is

enhancement of toxicity in the presence of an insecticide synergist that compromises the enzyme

in question (Feyereisen 2011). Suppression of resistance or reduction in levels of resistance by

the synergist, as measured by increased mortality, indicates involvement of the particular enzyme

system inhibited by the synergist in insecticide metabolism. In this experiment, the synergists

piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) were assessed in

bioassays with the pyrethroids bifenthrin and β-cyfluthrin in order to determine if P450s or

COEs contribute to pyrethroid resistance in a field-derived population of navel orangeworm from

Kern County, California. A susceptible laboratory colony of navel orangeworm was used to

compare the effects of the synergists and to calculate resistance level differences. Finally,

chlorantraniliprole and methoxyfenozide, in combination with bifenthrin and β-cyfluthrin, were

added to artificial diets to determine if insecticide tank mixtures could be effective in

overcoming resistance in this species.


Chemicals:

Piperonyl butoxide was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). β-

cyfluthrin, chlorantraniliprole, and methoxyfenozide were purchased from Sigma (St. Louis,

MO). Bifenthrin and S,S,S-tributyl phosphorotrithioate were purchased from Chem Service, Inc.

(West Chester, PA). Bifenthrin, and methoxyfenozide were dissolved in methanol and β-

cyfluthrin and chlorantraniliprole were dissolved in acetone. All stock solutions were stored at -

20ºC.

30

Navel orangeworm colonies:

A susceptible colony of A. transitella designated as CPQ (J. Siegel, USDA-ARS, Parlier,

CA) and a resistant colony designated as R347 (B. Higbee, Paramount Farming Company,

Bakersfield, CA) were kept in an incubator at University of Illinois at Urbana-Champaign and

maintained at conditions of 27 ± 4ºC in the absence of a light cycle. Larvae were mass-reared

until pupation in 500-ml glass jars containing 300 g of a wheat bran-based artificial diet (Finney

and Brinkmann 1967). Adults were transferred to additional 500-ml glass jars with tissue paper

on the inside and covering the top to serve as an oviposition substrate. Eggs were collected every

48 hours from these jars. First instar larvae were selected within 24 hours of egg hatch. Four

larvae were gently transferred into each 28-ml (1-oz) plastic cups containing 5 g of unaugmented

(control) diet or diet mixed with different concentrations of insecticide in the bioassays. A total

of 20 larvae (5 cups) were prepared for each treatment with or without insecticide application.

Insecticide Preparation:

Insecticides were incorporated into the artificial diet at specific concentrations and fed to

neonate larvae. Insecticides were mixed in with the diet in its liquid phase and then poured into

separate 28-ml (1-oz) cups to harden. Solutions of each pyrethroid in methanol or acetone were

prepared at a range of concentrations for each strain. The concentrations tested in the susceptible

strain were 50 ng/g, 200 ng/g, 300 ng/g, and 500 ng/g for bifenthrin and 50 ng/g, 100 ng/g, 150

ng/g, 500 ng/g, and 1 µ/g for β cyfluthrin. The concentrations tested in the resistant strain were

500 ng/g, 1 µg/g, 1.5 µg/g, 2 µg/g, and 4 µg/g for bifenthrin and 50 ng/g, 100 ng/g, 500 ng/g, 1

µg/g, and 2 µ/g for β cyfluthrin. In each bioassay, 20 neonates were exposed to each

concentration of insecticide. Mortality levels were assessed and recorded at each concentration

31

after 48 hours. Bioassays were replicated three times. Larvae that did not move when touched

with a soft brush were scored as dead.

Insecticides selected for use in tank mix assays with pyrethroids were prepared along a

range of concentrations and fed to neonate larvae (methoxyfenozide: 20 ng/g, 76 ng/g, 130 ng/g,

and 250 ng/g; chlorantraniliprole: 1 µg/g, 2 µg/g, 4 µg/g, and 8 µg/g). A 130 ng/g dose of

methoxyfenozide and 4 µg/g dose of chlorantraniliprole were selected based on mortality after

48 hours (~20%) for use in bioassays with the LC50 doses of bifenthrin and β-cyfluthrin.

Synergist Preparation:

A concentration of 200 µg/g for piperonyl butoxide was previously established by Niu et

al. (2012) as the maximum concentration determined to be nonlethal for first instar larvae. For

S,S,S-tributyl phosphorotrithioate, a concentration of 100 µg/g produced <15% morality after 48

hours, and this concentration was used in all assays.

Bioassays:

Median-lethal concentrations determined for β-cyfluthrin and bifenthrin within each

strain of navel orangeworms were mixed into the artificial diet in the presence or absence of

piperonyl butoxide or S,S,S-tributyl phosphorotrithioate. Controls in each bioassay included a

diet treatment in which the synergists PBO and DEF were mixed in with diet in the absence of

insecticide for the susceptible strain. The synergist DEF was selected as the only control for

pyrethroid assays with the resistant strain. Each bioassay in the susceptible strain involved 20

first instar larvae exposed to a single concentration of insecticide with or without synergist and

was replicated four times. Lower egg production from the resistant strain resulted in fewer

replicates, fewer individuals per replicate, and the absence of a PBO control. Bioassays in the

resistant strain involved 15 first instar larvae exposed to a single concentration of insecticide

32

with or without synergist and were replicated four times. Bioassays involving insecticide mixes

were administered with 15 first instar larvae per treatment and replicated three times. Controls in

the insecticide mix assays consisted of 200 µl insecticide solvent mixed into diet in the absence

of insecticide. On the rare occasions that cannibalistic behavior or scavenging on already-dead

caterpillars occurred, larvae were removed from the sample size.


SPSS version 22 software (SPSS Inc., Chicago IL) was used to run the Probit analysis

and generate an estimated calculation of the median lethal concentration that would kill 50% of

the sample population at 48 hours (LC50) for the pyrethroids. The median lethal concentration of

each insecticide was set as a baseline, and synergistic were observed when the mortality of the

insecticide with synergist was significantly higher (P<0.05) than the mortality of the insecticide

alone. Separate analyses using JMP Pro version 9 (SAS Institute, Cary NC) were calculated for

each insecticide and insecticide with synergist using multiple regression with orthogonal

polynomial contrasts in order to differentiate among treatments.

Results

The LC50 values for bifenthrin and β-cyfluthrin for the resistant and susceptible strains

are significantly different as shown by their non-overlapping 95% confidence intervals (Fig. 2.1).

The LC50 was on average 8.7 -fold greater (ranging from 6.2 to 11.7-fold) in the resistant strain

than the susceptible strain for bifenthrin, and the LC50 was 11-fold greater (ranging from 4.7 to

17.8-fold) in the resistant strain than the susceptible strain for β-cyfluthrin.

In the susceptible strain, mortality levels were significantly different (P<0.001) across all

time points examined between insecticide treatments and the control in bifenthrin assays. There

was no significant difference observed between PBO and DEF (P>0.05) at any time point

33

examined. Both PBO and DEF synergized the toxicity of bifenthrin at 72 hours (P<0.001)

through 144 hours (P<0.001) (Fig. 2.2). The treatments including either PBO or DEF with

bifenthrin were not significantly different from each other after 144 hours (P=0.154). Mortality

levels were significantly different (P<0.001) across all time points between insecticide

treatments and the control in β-cyfluthrin assays. PBO and DEF synergized the toxicity of β-

cyfluthrin at 72 hours (P<0.001) through 144 hours (P<0.001) (Fig. 2.3). Treatments including

either PBO or DEF with β-cyfluthrin were significantly different from each other at 72 hours

(P=0.005) through 144 hours (P=0.035).

In the resistant strain, mortality levels were significantly different (P<0.001) across all

time points between insecticide treatments and the control in bifenthrin assays. Both PBO and

DEF synergized the toxicity of bifenthrin at 72 hours (P<0.001) through 144 hours (P<0.001)

(Fig. 2.4). Treatments including either PBO or DEF with bifenthrin were not significantly

different from each other after 144 hours (P=0.136). Mortality levels were significantly different

(P<0.001) across all time points between insecticide treatments and the control in β-cyfluthrin

assays. Both PBO and DEF synergized the toxicity of β-cyfluthrin at 48 hours (P<0.001) through

144 hours (P<0.001) (Fig. 2.5). Treatments including either PBO or DEF with β-cyfluthrin were

not significantly different from each other after 144 hours (P=0.486)

In bioassays aimed at determining the effect of methoxyfenozide on toxicity of

pyrethroids, mortality levels were significantly different (P<0.001) across all time points

between insecticide treatments and the control for bioassays that combined bifenthrin and β-

cyfluthrin with methoxyfenozide. Methoxyfenozide had no significant effect on the toxicity of

either bifenthrin in mixtures (Fig. 2.6) from 48 hours (P=0.106) to 72 hours (P=0.459) or β-

cyfluthrin in mixtures (Fig. 2.7) from 48 hours (P=0.831) to 72 hours (P=0.355). The toxicity of

34

methoxyfenozide was significantly different from the toxicity of bifenthrin at 48 hours (P<0.001)

but not 72 hours (P=0.222). The toxicity of methoxyfenozide was significantly different from the

toxicity of β-cyfluthrin at 48 hours (P<0.001) but not 72 hours (P=0.182).

In bioassays aimed at determining the effect of chlorantraniliprole on toxicity of

pyrethroids, mortality levels were significantly different (P<0.001) across all time points

between insecticide treatments and the control for bioassays that combined bifenthrin and β-

cyfluthrin with chlorantraniliprole. Chlorantraniliprole significantly increased the toxicity of

bifenthrin in mixtures (Fig. 2.8) at 72 hours (P=0.003) but not 48 hours (P=0.196).

Chlorantraniliprole increased the toxicity of β-cyfluthrin in mixtures (Fig. 2.9) at 48 hours

(P=0.015) and (P<0.001) at 72 hours. The toxicity of chlorantraniliprole was significantly

different from the toxicity of bifenthrin after 48 hours (P=0.045) but not 72 hours. (P=0.179).

The toxicity of chlorantraniliprole was significantly different from the toxicity of β-cyfluthrin

after 48 hours (P=0.010) but not 72 hours (P=0.592).

Discussion

Suppression of resistance in vivo in bioassays that incorporate inhibitors such as PBO or

DEF accentuates the contributions of P450s and COEs in the detoxification of pyrethroids

(Oakeshott et al. 2005), as opposed to target-site resistance that is usually inferred when insects

display no change in pesticide sensitivity in the presence of such synergists (Khambay and

Jewess 2005). As expected, applications of PBO and DEF synergized the toxicity of bifenthrin

and β-cyfluthrin in the susceptible strain of navel orangeworms, confirming that P450s and

COEs are involved in detoxification in this strain as well. Results from our bioassays show that

the same synergistic concentrations of PBO and DEF in the susceptible strain decreased the LC50

35

for bifenthrin and β-cyfluthrin in the resistant strain. Resistance is therefore at least partly

metabolic and likely occurs through overexpression of specific P450 and COE genes.

Recent studies involving highly polyphagous herbivorous pests such as the corn earworm

(Helicoverpa zea) (Lepidoptera: Noctuidae) suggest that P450s responsible for insecticide

detoxification may have evolved from P450s that detoxify host plant allelochemicals (Li et al.

2007). In H. zea, CYP321A1 and CYP6B8 are involved in the metabolism of the pyrethroid

cypermethrin as well as many phytochemicals (Li et al. 2004; Sasabe et al. 2004). The use of

pyrethroid insecticides to control the Old World bollworm (Helicoverpa amigera) (Lepidoptera:

Noctuidae) has resulted in populations that owe their resistance to overexpression of

detoxificative P450s (Martin et al. 2002). In navel orangeworm, CYP6AB11 metabolizes

imperatorin, a furanocoumarin related to those found in figs and citrus species (Niu et al. 2011).

Whether P450s that are specialized for metabolizing a narrow range of alleochemicals in the

broadly polyphagous navel orangeworm contribute to development of resistance to pyrethroids is

an open question, but the fact that synthetic pyrethroids are structurally derived from plant

alleochemicals makes such a scenario plausible (Li et al. 2007).

β-cyfluthrin is classified as a type II pyrethroid because it contains an α-cyano group at

its ester linkage (Davies et al. 2007). The presence of the α-cyano group makes pyrethroids less

susceptible to hydrolytic detoxification by carboxylesterases (Casida et al. 1983; Oakeshott et al.

2005). β-cyfluthrin detoxification differed between the resistant and the susceptible strains

because the magnitude of the effect of the COE inhibitor was significantly less than the effect of

the P450 inhibitor in the susceptible strain. In the resistant strain, the inhibitors had the same

effect on the LC50, which may implicate equal contributions from P450s and COEs in the

detoxification of β-cyfluthrin, but results may be confounded by the fact that PBO is also a

36

moderate inhibitor of esterases in some insects, and DEF has some capacity to inhibit P450 genes

in some insects (Ahmad and Hollingsworth 2004).

Cross-resistance is a major problem in designing management strategies once an insect

pest has acquired resistance to a particular insecticide (Liu et al. 2012). If resistance to one

insecticide class can emerge as the result of enhanced P450 and COE activity, then cross-

resistance may arise at an increasing rate if resistant navel orangeworms are exposed to other

insecticides that share similar modes of detoxification. I evaluated the efficacy of

methoxyfenozide and chlorantraniliprole, which are combined in tank mixes with pyrethroids

that are registered for use in almond and pistachio orchards.

Niu et al. (2012) determined that methoxyfenozide is not detoxified by P450s in a

susceptible strain of navel orangeworms. Methoxyfenozide did not synergize or inhibit the

toxicity of bifenthrin or β-cyfluthrin in the resistant strain of navel orangeworms. Pyrethroids and

methoxyfenozide in mixes may be detoxified through different pathways in the navel

orangeworm, but, based on the results of our bioassays, methoxyfenozide would not be expected

to effectively restore susceptibility in resistant strains through synergistic effects.

In contrast, chlorantraniliprole synergized the toxicity of bifenthrin and β-cyfluthrin.

Although its main detoxification pathway has yet to be determined in the navel orangeworm, our

results suggest that chlorantraniliprole can be used in combination with pyrethroids to

circumvent resistance because of its ability to synergize combined with an independent mode of

detoxification. Future assays that involve combinations of pesticides already registered for use

against navel orangeworm can provide insight into whether mixtures containing

chlorantraniliprole can be effective in overcoming existing pyrethroid resistance.

37

The proximity between almond and pistachio orchards in California’s Central Valley

could have a major impact on the dispersal and establishment of resistant populations. Higbee

and Siegel (2009) assessed navel orangeworm damage patterns in almonds and found

significantly more damage in nuts harvested within 3 miles (4.8 km) of pistachio orchards

compared to those farther away. Studies in 2003 and 2004 (Burks et al. 2008) showed that

pistachio orchards in Kern County contained denser populations of navel orangeworms than in

almond orchards. Sanitation is more difficult to accomplish in pistachio orchards than in almond

orchards because pistachios cannot be readily shredded by tillage, which results in a greater

abundance of mummies and thus overwintering sites for the navel orangeworm (Siegel et al

2008). The proximity of these other hosts and the different timing of insecticide use in these

crops also increase the possibility of nontarget exposure of navel orangeworms to sublethal

insecticide concentrations, which could accelerate resistance acquisition and threaten integrated

management plans.

38

Figures

Figure 2.1. Median-lethal concentration (LC50) values after 48 hours for the pyrethroid

insecticides bifenthrin and β-cyfluthrin. Error bars represent the 95% confidence limits in the

LC50 for each insecticide

0

0.5

1

1.5

2

2.5

Bifenthrin β-cyfluthrin

Do

se (

µg

/g)

Pyrethroid

LC50 after 48 hours

Susceptible strain

Resistant strain

39

Figure 2.2. First instar navel orangeworm (CPQ) mortality across multiple time points following

dietary exposure to 200 ng/g bifenthrin, 200 ng/g bifenthrin +200 µg/g piperonyl butoxide

(PBO), 200 ng/g bifenthrin + 100 µg/g S,S,S-tributyl phosphorotrithioate (DEF), 200 µg/g PBO,

and 100 µg/g DEF. Error bars represent the standard error. Letters A, B, and C represent


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


PBO

DEF

Bifenthrin

Bifenthrin + PBO

Bifenthrin + DEF

A

A

B

C

C

40

Figure 2.3. First instar navel orangeworm (CPQ) mortality across multiple time points following

dietary exposure to 40 ng/g β-cyfluthrin, 40 ng/g β-cyfluthrin + 200 µg/g piperonyl butoxide

(PBO), 40 ng/g β-cyfluthrin + 100 µg/g S,S,S-tributyl phosphorotrithioate, 200 µg/g PBO, and

100 µg/g DEF. Error bars represent the standard error. Letters A, B, C, and D represent


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


PBO

DEF

β-cyfluthrin

β-cyfluthrin + PBO

β-cyfluthrin + DEF

A

B

C

D

D

41

Figure 2.4. First instar navel orangeworm (R347) mortality across multiple time points

following dietary exposure to 1.6 µg/g bifenthrin, 1.6 µg/g bifenthrin + 200 µg/g piperonyl

butoxide (PBO), 1.6 µg/g bifenthrin + 100 µg/g S,S,S-tributyl phosphorotrithioate (DEF), and

100 µg/g DEF. Error bars represent the standard error. Letters A, B, and C represent significantly

different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


DEF

Bifenthrin

Bifenthrin + PBO

Bifenthrin + DEF

A

A

B

C

42


following dietary exposure to 350 ng/g β-cyfluthrin, 350 ng/g β-cyfluthrin + 200 µg/g piperonyl

butoxide (PBO), 350 ng/g β-cyfluthrin + 100 µg/g S,S,S-tributyl phosphorotrithioate (DEF), and

100 µg/g DEF. Error bars represent the standard error. Letters A, B, and C represent significantly

different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


DEF

β-cyfluthrin

β-cyfluthrin + PBO

β-cyfluthrin + DEF

A

A

B

C

43


following dietary exposure to 1.6 µg/g bifenthrin, 130 ng/g methoxyfenozide, 1.6 µg/g bifenthrin

+ 130 ng/g methoxyfenozide, and a control (200 µl methanol). Error bars represent the standard

error. Letters represent significantly different groups (P<0.05). Letters A and B represent


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Methanol

Bifenthrin

Methoxyfenozide

Bifenthrin +

Methoxyfenozide

A A A

B

44


following dietary exposure to 350 ng/g β-cyfluthrin, 130 ng/g methoxyfenozide, 350 ng/g β-

cyfluthrin + 130 ng/g methoxyfenozide, and a control (200 µl methanol). Error bars represent the

standard error. Letters A and B represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Methanol

β-cyfluthrin

Methoxyfenozide

β-cyluthrin +

Methoxyfenozide

A

A

A

B

45


following dietary exposure to 1.6 µg/g bifenthrin, 4 µg/g chlorantraniliprole, 1.6 µg/g bifenthrin

+ 4 µ/g chlorantraniliprole, and a control (200 µl acetone). Error bars represent the standard

error. Letters A, B, and C represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Acetone

Bifenthrin

Chlorantraniliprole

Bifenthrin +

Chlorantraniliprole

A

B

C

B

46


following dietary exposure to 350 ng/g β-cyfluthrin, 4 µg/g chlorantraniliprole, 350 ng/g β-

cyfluthrin + 4 µ/g chlorantraniliprole, and a control (200 µl acetone). Error bars represent the

standard error. Letters A, B, and C represent significantly different groups (P<0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120 144

Mo

rta

lity


Acetone

β-cyfluthrin

Chlorantraniliprole

β-cyfluthrin +

Chlorantraniliprole

A

B

B

C

47

References

[ACP] Administrative Committee for Pistachios Processors' Producer Delivery Reports and

Acreage Surveys. 2011 Pistachio Bearing Acreage Report. http://www.acpistachios.org.

Ahmad, M., and Hollingworth, R.M. (2004) Synergism of insecticides provides evidence of

metabolic mechanisms of resistance in the obliquebanded leafroller Choristoneura

rosaceana (Lepidoptera: Tortricidae). Pest Manag. Sci. 60: 465-473.



pests of pistachio. Pistachio Production Manual: 179-191.

Burks, C.S., Higbee, B.S., Brandl, D.G., and Mackey, B.E. (2008) Sampling and pheromone

trapping for comparison of abundance of Amyelois transitella in almonds and

pistachios. Entomol. Exp. Appl. 129: 66-76.

Casida, J.E., Gammon, D.W., Glickman, A.H., and Lawrence, L.J. (1983) Mechanisms of

selective action of pyrethroid insecticides. Annu. Rev. Pharmacol. Toxicol. 23: 413-438.

Connell, J.H. (2001) Leading edge of plant protection for almond. Hortic. Technol. 12: 619–622.

[CPRB] California Pistachio Research Board. 2012 Pistachio Pesticides.

www.calpistachioresearch.org/PISTACHIO_PESTICIDES-_insecticides.pdf.

Davies, T.G.E., Field, L.M., Usherwood, P.N.R., and Williamson, M.S. (2007). DDT, pyrethrins,

pyrethroids and insect sodium channels. IUBMB Life 59: 151-162.

Feyereisen, R. (2005) Insect cytochrome P450. In: Gilbert, L.I., Latrou, K., Gill, S.S. (eds)

Comprehensive molecular insect science, vol. 4, chapter 1. Elsevier, Oxford, pp 1–77.

Feyereisen, R (2011) Insect CYP genes and P450 enzymes. Insect Molecular Biology and

Biochemistry 450: 236–316. Oxford, UK: Elsevier.

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Finney, G.L., and Brinkmann, D. (1967) Rearing the navel orangeworm in the laboratory. J.

Econ. Entomol. 60: 1109-1111.

Gunning, R.V., Easton, C.S., Balfe, M.E. and Ferris, I.G. (1991) Pyrethroid resistance

mechanisms in Australian Helicoverpa armigera. Pestic. Sci. 33: 473–490.

Higbee, B.S. and Siegel, J.P. (2009) New navel orangeworm sanitation standards could reduce

almond damage. Calif. Agric. 63: 24-28.


eds Gilbert, L.I., Iatrou, K., and Gill, S.S. (Elsevier, Oxford), vol. 6, pp. 1–29..

Lee, S.E., and Campbell, B.C. (2000) In vitro metabolism of aflatoxin B1 by larvae of navel

orangeworm, Amyelois transitella (Walker) (Insecta, Lepidoptera, Pyralidae) and codling

moth, Cydia pomonella (L.) (Insecta, Lepidoptera, Tortricidae). Arch. Insect Biochem.

Physiol. 45: 166–174.

Li, X.C., Baudry, J., Berenbaum, M.R. and Schuler, M.A. (2004) Structural and functional

divergence of insect CYP6B proteins: from specialist to generalist cytochrome

P450. Proc. Natl. Acad. Sci. USA 101: 2939–2944.



Liu, N. (2012). Pyrethroid resistance in insects: genes, mechanisms, and regulation. Insecticides

advances in integrated pest management (ed. F. Perveen) 457-468.

Martin, T., Chandre, F., Ochou, O.G., Vaissayre, M., and Fournier, D. (2002) Pyrethroid

resistance mechanisms in the cotton bollworm Helicoverpa armigera Lepidoptera:

Noctuidae) from West Africa. Pestic. Biochem. Physiol. 74: 17-26.

49

[NASS] National Agricultural Statistics Service. 2005 Almond Acreage Report 2006.

Sacramento, CA:US Department of Agriculture. 8p. www.nass.usda.gov.

Niu, G., Rupasinghe, S.G., Zangerl, A.R., Siegel, J.P., Schuler, M.A., and Berenbaum, M.R.

(2011) A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the

polyphagous navel orangeworm (Amyelois transitella). Insect Biochem. Mol. Biol. 41:

244–253.

Niu, G., Pollock, H., Lawrance, A., Siegel, J., and Berenbaum, M.R. (2012) Effects of a naturally

occurring and a synthetic synergist on toxicity of three insecticides and a phytochemical

to navel orangeworm (Lepidoptera: Pyralidae). J. Econ. Entomol. 105: 410-417.

Oakeshott, J.G., Claudianos, C., Campbell, P.M., Newcomb, R.D., and Russell, R.J. (2005)

Biochemical genetics and genomics of insect esterases. In: Gilbert, L.I., Iatrou, K, Gill


Ranson, H., and Hemingway, J. (2005) Glutathione transferases. In: Gilbert, L.I., Iatrou, K., Gill,


Sasabe, M., Wen, Z., Berenbaum, M.R., Schuler, M.A. (2004) Molecular analysis ofCYP321A1,

a novel cytochrome P450 involved in metabolism of plant allelochemicals

(furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea. Gene 388: 163–

175.

Siegel, J.P., Higbee, B.S., Noble, P., Gill, R., Yokota, G.Y., Krugner, R., & Daane, K.M. (2008).

Postharvest survival of navel orangeworm assessed in pistachios. Calif. Agric. 62: 30-35.




50

Zalom, F.G., Pickle, C., Bentley, W.J., Haviland, D.R., Van Steenwyk, R.A., Rice, R.E.,

Hendricks, L.C., Coviello, R.L., and Freeman, M.W. (2012) UC IPM Pest Management

Guidelines: Almond. University of California ANR Publication 3431.

51

CHAPTER III: LIFE HISTORY DIFFERENCES BETWEEN A RESISTANT AND

SUSCEPTIBLE STRAIN OF NAVEL ORANGEWORMS

Introduction

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the

primary pest of almonds and pistachios in California orchards (Siegel et al. 2010). The navel

orangeworm is managed primarily through winter removal and destruction of mummies along

with insecticide use. Navel orangeworm populations undergo two generations on mummies

before adults emerge and oviposit on new crop nuts during hull split from late July to August

(Sanderson et al. 1989; Bentley et al. 2008). Insecticides that target the most damaging third

generation may be timed based on oviposition activity or onset of crop susceptibility (Sanderson

et al 1989). Pyrethroid insecticides and insect growth regulators are the insecticides most widely

used during the growing season (Leal et al. 2009).

Resistance to the pyrethroid insecticide bifenthrin has recently been reported in almond

orchards located in Kern County. Insect pests can acquire resistance to pyrethroid insecticides

through multiple mechanisms, including as target-site insensitivity, overexpression of essential

detoxification enzymes, increased sequestration, and reduced cuticular penetration (Khambay

and Jewess 2005; Li et al. 2007; Liu 2012). Our previous study (Chapter 2) demonstrated that the

mechanism of resistance in this resistant population involves elevated levels of cytochrome P450

monooxygenase (P450) and carboxylesterase (COE) activity.

The acquisition of resistance to synthetic insecticides often carries an associated fitness

cost. A reduction in fitness occurs in resistant populations when resources directed toward

fitness-enhancing traits are diverted in favor of the mechanisms involved in the production and

maintenance of resistance (Carriere et al. 1994). Costs associated with insecticide resistance such

52

as increased development time of larval and pupal stages have been observed in many

lepidopteran pests, including the tobacco budworm (Heliothis virescens) (Lepidoptera:

Noctuidae) (Sayyed et al. 2008), the cotton bollworm (Helicoverpa armigera) (Lepidoptera:

Noctuidae) (Wang et al. 2010), and the obliquebanded leafroller (Choristoneura rosaceana)

(Harris) (Carriere et al. 1994).

In this study, I compared life history traits in a resistant strain of navel orangeworms and

a susceptible strain in an effort to identify fitness costs associated with the initial development of

resistance to pyrethroid insecticides. Life tables offer an effective means of summarizing the

survival and mortality rates in a population and allow for statistical inferences about the

population under study (Chiang 1984). I constructed life tables from first instar through pupation

for resistant and susceptible strains on a semi-synthetic artificial diet (Niu et al. 2012). Overall

survivorship, development time, and pupal weights were compared between strains. In addition,

pupal weights were compared directly across three generations from resistant and susceptible

strains reared on wheat bran diet. Median-lethal concentrations (LC50) for bifenthrin were also

recorded through eight generations to determine if resistance was maintained in the absence of

bifenthrin selection pressure.


Insects:

A laboratory colony of A. transitella designated as CPQ (J. Siegel: USDA-ARS, Parlier,

CA) and a resistant colony designated as R347 (B. Higbee, Paramount Farming Company,

Bakersfield, CA) were kept an incubator at University of Illinois at Urbana-Champaign and

maintained at conditions of 27 ± 4ºC in the absence of a light cycle. Larvae were mass-reared

until pupation in 500 ml glass jars containing 300 g of a wheat bran diet (Finney and Brinkmann

53

1967). Adults were transferred to additional 500-ml glass jars with tissue paper on the inside and

covering the top to serve as oviposition substrate. Eggs were collected every 48 hours from these

jars.

Life Table:

Eggs were collected on the same day for both the susceptible and the resistant colonies.

After egg hatch, 50 neonates from each strain were transferred individually onto separate 28-ml

(1-oz) plastic cups containing 5 g of artificial diet. Larvae were checked every day for the

occurrence of a molt and for survivorship through the first five larval instars prior to pupation.

The presence of a shed head capsule indicated a molt and a transition to the next stadium. Days

when molting occurred were recorded until the larvae pupated. Individuals that survived to

pupation were removed, separated by sex, and weighed for comparison between strains. Life

tables were generated based on the percentage survivorship at the beginning of each stage (lx),

the number dying during each stage (dx), and the mortality rate between instars (qx) (Table 3.1).

Boxplots were constructed to examine the distribution of days until pupation and the first molt

from individuals in the susceptible and resistant strains The diet was replaced once each week in

order to avoid any effects of spoilage throughout the experiment.

Pupal Weights:

Pupal weights of at least 200 individuals were randomly sampled in each generation

from the susceptible and resistant colonies reared on the wheat bran diet. Pupae from each strain

were separated by sex within each generation and the number of males and females was

recorded. The sex of a pupa was determined by the location of the gonopore, which occurs on the

eighth segment in females and ninth segment in males (Husseiny and Madsen 1964).

54

Median-lethal Concentration Assays:

Bifenthrin was mixed into the artificial diet during the liquid phase at specific

concentrations and poured into separate 1 oz (28 ml) cups to harden. Solutions of bifenthrin in

methanol were prepared at a range of concentrations (500 ng/g, 1 µg/g, 2 µg/g, 3 µg/g, and 8

µg/g) and fed to neonate larvae. For each bioassay, 20 neonates were exposed to each

concentration of bifenthrin. Mortality levels were assessed and recorded at each concentration

after 48 hours. Larvae that did not move when touched with a soft brush were scored as dead.

Bioassays were repeated twice in the second and third generations and three times in generations

three through eight.


SPSS version 22 software (SPSS Inc., Chicago IL) was used to construct Kaplan-Meier

(1958) curves and test for a significant difference in survivorship across five larval instars and

through pupation in the resistant and susceptible strains. A two-tailed t-test was used in SPSS to

check for significant differences between pupal weights and development times of both strains

from life table experiments. A two-way analysis of variance (ANOVA) was run in SPSS to test

for significant differences in pupal weights across multiple generations for females and males of

each strain that were reared on wheat bran diet. Strain and generation were the categorical

variables in the ANOVA. Finally, the Probit analysis was used to calculate median-lethal

concentrations that would kill 50% of resistant neonates at 48 hours for the insecticide bifenthrin.

Results

Kaplan-Meier survivorship curves were constructed to determine if the difference in

survivorship between the susceptible strain and the resistant strain was significant from first

instar through pupation (Fig. 3.1). Individuals that survived to pupation were marked as censored

55

and removed from the analysis. One individual present from each strain in the fifth instar was

also censored after 60 days (indicative of developmental abnormalities) since the start of the

experiment. The Log-Rank Test indicated that survivorship was not significantly different

between the susceptible strain and the resistant strain (P=0.146).

Results from the t-test confirmed there was no significant difference in time to pupation

between strains (P=0.732) (Fig. 3.2). The boxplot of time to the first molt displayed two outliers

at 9 days and 14 days in the distribution from the resistant strain. These outliers were removed in

the two-tailed t-test, which concluded that the difference in the number of days until the molt

into second instar between strains was significant (P<0.001) (Fig. 3.3).

The t-test confirmed there was no significant difference in weight of female pupae from

the susceptible and resistant strains, although there was a trend toward larger weights in the

susceptible strain (P=0.062) (Fig. 3.4). There was no significant difference in weight of male

pupae from the susceptible and resistant strains (P=0.333). The effects of strain (P<0.001) and

generation (P<0.001) were significant in the two-way ANOVA for females, while the interaction

was not significant (P=0.081) (Fig. 3.5). In males, the effects of strain (P<0.001) and generation

(P<0.001) were significant in the two-way ANOVA as was the interaction (P<0.001) (Fig. 3.6).

Separate one-way ANOVAs were conducted between males from the susceptible and resistant

strains in generations 4, 5, and 6. Results from the one-way ANOVAs were significant between

strains for each generation of males (P<0.001).

The goodness-of-fit test indicated that bifenthrin concentration mortality data fit the

Probit model (P>0.05) in each of the eight generations (Table 3.2). The LC50 values for

bifenthrin in the resistant strain across eight generations were not considered significantly

different as evidenced by their non-overlapping 95% confidence intervals.

56

Discussion

Although genetically determined resistance may provide a selective advantage to

individuals in the presence of an insecticide, resistance genes are rarely fixed in natural

populations and can be lost in the absence of selection pressure exerted by insecticides (Coustau

and Chevillon 2000). If insecticide resistance carries a strong associated fitness cost, then a

return to an insecticide-free environment could result in a strong phenotypic change with

deleterious effects (Coustau and Chevillon 2000; Kliot and Ghanim 2012). Boivin et al. (2003)

reported a decline in laboratory-reared codling moths (Cydia pomonella) (Lepidoptera:

Tortricidae) resistant to the benzamide insecticide diflubenzuron after 10 generations in the

absence of insecticide pressure. Because navel orangeworm resistance to bifenthrin developed

recently, it is possible that alleles underlying enhanced detoxification have not become fixed

within the population and may be lost in the absence of bifenthrin selection pressure. Median-

lethal concentration assays for bifenthrin over first 8 generations under laboratory conditions

indicate resistance is heritable thus far and that contributing alleles are maintained in the resistant

colony.

Initial differences in development of the resistant strain compared with the susceptible

strain could have an impact on management practices for navel orangeworm. The increased

mortality displayed in the initial stages of growth from the life table experiment and the

significant difference in the number of days to the first molt could influence efficacy of

insecticide sprays. If resistant strains develop more slowly during the initial stages of growth in

almond orchards, then populations may experience greater exposure and impact from

allelochemicals present in the shells and hulls of almonds in addition to prolonged insecticide

treatments before they can tunnel into the nut (Siegel et al. 2010).

57

The recorded survival and development across all larval stages and through pupation,

however, did not support the existence of fitness costs associated with development in the

resistant strain. Control failure in navel orangeworm populations may result when variation in

development times produces faster-growing individuals, potentially resulting in an extra

generation or generations that substantially increases the standing population; alternatively,

individuals that develop more slowly may emerge and oviposit on new-crop nuts after insecticide

treatment has degraded (Siegel et al. 2010). If there is no effect on the development of navel

orangeworm populations resistant to pyrethroids, then the timing of insecticide sprays that target

adult oviposition during the spring and at hull split may not result in deviations from standard

management practices.

The positive relationship between female pupal weight and fecundity has been

documented in many species of Lepidoptera (Haukioja and Neuvonen 1985; Calvo and Molina

2005; Konopka et al. 2012). Comparisons of pupal weights across three generations revealed that

females were larger in the susceptible strain than the resistant strain. If these observed

differences are reflected in resistant field populations, then the dispersal and establishment of

additional resistant populations could be severely hindered by reduced fecundity associated with

smaller adult size.

Pupal weight comparisons across three generations in males also indicated a potential

fitness cost in the resistant strain through significant reductions in size. Male size can have a

direct impact on ejaculate size and nutritional content in spermatophores (Wiklund and Kaitala

1995). In the male European corn borer (Ostrinia nubilalis) (Lepidopetera: Crambidae), the

volume of the first spermatophore produced is correlated with pupal weight, and investments in

the production of sperm and accessory gland secretions may limit reproductive outputs (Royer

58

and McNeil 1993). Pyrethroid resistance in navel orangeworm populations may limit the number

of offspring males can produce if smaller size has an impact on the quality of spermatophores in

field populations.

Identifying fitness costs resulting from pyrethroids can be extremely valuable in

designing pest management programs that can limit the spread of the resistant population.

Results from this study suggest a fitness cost associated with pyrethroid resistance, but such

assessments conducted under optimal laboratory conditions may not accurately reflect the

conditions experienced by field populations of navel orangeworms (Kliot and Ghanim 2012).

Pupal weight differences may simply reflect heterogeneity in the resistant strain or initial

adaptation to the laboratory conditions as opposed to fitness costs. It is possible that differences

noted in the resistant strain may have resulted from suites of alleles maintained or lost through

founder effects or drift. Differences in weights between susceptible and resistant lines are

confounded by a separate origin for the two lines, combined with a large difference in the

number of generations in laboratory culture. Examining additional navel orangeworm

populations in the orchards of Kern and its neighboring counties will be invaluable in

determining the distribution of resistant individuals and its association with different histories of

pyrethroid use.

59

Figures and Tables

Figure 3.1. Kaplan-Meier Survivorship Curves for a resistant (R347) and susceptible strain

(CPQ) of Amyelois transitella. The sixth instar on the X-axis represents the pupal stage.

Strain

Resistant

Susceptible

Resistant-Censored

Susceptible-Censored

60

Figure 3.2. Distribution of days to pupation for a resistant (R347) and susceptible strain (CPQ)

of A. transitella from a life table experiment. Error bars (whiskers) represent the highest and

lowest values below 1.5 and 3 times the interquartile range. Circles represent outliers.

n=29 n=35

61

Figure 3.3. Distribution of days until first molt for a resistant (R347) and susceptible strain

(CPQ) of A. transitella from a life table experiment. Error bars (whiskers) represent the highest

and lowest values below 1.5 and 3 times the interquartile range. Circles represent outliers.

Outliers at 9 days and 14 days in the resistant strain were removed in the test for significance.

n=44 n=49

62

Figure 3.4. Pupal weights for male and female survivors during life table experiments in a

resistant (R347) and susceptible strain (CPQ) of A. transitella. Error bars represent standard

error.

0

10

20

30

40

50

60

Susceptible Resistant

Wei

gh

t (m

g)

Strain

Male

Female

n=23

n=13

n=16

n=12

63

Figure 3.5. Mean pupal weight of females reared on wheat bran diet from a resistant (R347) and

susceptible strain (CPQ) of A. transitella across multiple generations. Error bars represent the

standard error.

40

42

44

46

48

50

52

54

F2 F3 F4 F5 F6 F7 F8

Wei

gh

t (m

g)

Generation

Resistant ♀

Susceptible ♀ n=108

n=124

n=130

n=132

n=121

n=122

n=105

n=113 n=119 n=101 n=102

64

Figure 3.6. Mean pupal weight of males reared on wheat bran diet from a resistant (R347) and

susceptible strain (CPQ) of A. transitella across multiple generations. Error bars represent the

standard error.

30

32

34

36

38

40

42

44

F2 F3 F4 F5 F6 F7 F8

Wei

gh

t (m

g)

Generation

Resistant ♂

Susceptible ♂

n=108

n=101 n=95

n=101

n=83

n=100 n=97

n=130 n=103

n=83

n=102

65

Table 3.1. Life table for a resistant (R347) and susceptible strain (CPQ) of A. transitella.

x Strain n lx dx qx

Egg Susceptible 50 1 0 0

Resistant 50 1 0 0

1st instar Susceptible 50 1 0 0

Resistant 50 1 0 0

2nd instar Susceptible 49 0.98 1 0.02

Resistant 44 0.88 6 0.12

3rd instar Susceptible 44 0.88 5 0.102

Resistant 37 0.74 7 0.159

4th instar Susceptible 42 0.84 2 0.045

Resistant 35 0.7 2 0.054

5th instar Susceptible 40 0.8 2 0.048

Resistant 32 0.64 3 0.086

Pupae Susceptible 35 0.7 4 0.1

Resistant 29 0.58 2 0.063

66

Table 3.2. Probit analysis data for bifenthrin in resistant neonate A. transitella across eight

generations maintained under laboratory conditions.

Generation n Slope (SE) LC50 ± 95% CL (ppm) x2 P

F2 200 1.93 (0.23) 1.88 (1.36-2.46) 3.58 0.17

F3 200 1.32 (0.27) 1.47 (1.05-1.98) 0.35 0.84

F4 300 1.87 (0.29) 1.89 (1.53-2.38) 0.48 0.79

F5 300 1.69 (0.20) 1.75 (1.40-2.16) 1.82 0.61

F6 300 1.90 (0.31) 1.52 (1.08-1.94) 1.58 0.45

F7 300 2.14 (0.23) 1.52 (1.27-1.81) 1.26 0.74

F8 300 1.88 (0.22) 1.83 (1.50-2.22) 0.56 0.91

67

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INSECTICIDE DETOXIFICATION IN THE NAVEL ORANGEWORM